Spectrum of genetic variants in 306 patients with non-syndromic hearing loss from Croatia

Aim To determine the spectrum and frequency of disease-causing variants in patients with non-syndromic hearing loss (NSHL) and to investigate the diagnostic yield of the applied genetic methods. Methods The study enrolled 306 unrelated patients with childhood-onset, mild-to-profound NSHL referred to Children’s Hospital Zagreb for genetic testing between March 2006 and October 2023. The GJB2 variants were analyzed with the multiplex ligation-dependent probe amplification method and Sanger sequencing of the coding region of the GJB2 gene. In 21 patients negative for GJB2 biallelic variants, clinical exome sequencing (CES) was performed. Results Among 234 disease-associated GJB2 alleles detected, 19 were clinically relevant, of which 18 were reported as pathogenic/likely pathogenic. The c.35delG variant accounted for 73.5% of the mutated alleles. More than half of the patients with biallelic GJB2 variants (64/110, 58.2%) were 35delG homozygotes. Seventeen non-GJB2 variants were found in 10 genes (TECTA, NOG, SLC26A4, PCDH15, TMPRSS3, USH2A, GATA3, MYO15A, SOX10, COL2A1) in 11 participants, and 5 variants (in TECTA, NOG, PCDH15, and SOX10) were novel (29.4%). Conclusion We were able to elucidate the genetic cause of hearing loss in 121 patients, with an overall diagnostic rate of 39.5%. The c.35delG was the most common variant. CES allowed us to diagnose almost half of the patients with HL; to distinguish NSHL from the syndromic form of HL in cases where the phenotype was unclear or where symptoms were absent from an early age; and to discover novel variants.


Aim
To determine the spectrum and frequency of diseasecausing variants in patients with non-syndromic hearing loss (NSHL) and to investigate the diagnostic yield of the applied genetic methods.

Methods
The study enrolled 306 unrelated patients with childhood-onset, mild-to-profound NSHL referred to Children's Hospital Zagreb for genetic testing between March 2006 and October 2023.The GJB2 variants were analyzed with the multiplex ligation-dependent probe amplification method and Sanger sequencing of the coding region of the GJB2 gene.In 21 patients negative for GJB2 biallelic variants, clinical exome sequencing (CES) was performed.

Conclusion
We were able to elucidate the genetic cause of hearing loss in 121 patients, with an overall diagnostic rate of 39.5%.The c.35delG was the most common variant.CES allowed us to diagnose almost half of the patients with HL; to distinguish NSHL from the syndromic form of HL in cases where the phenotype was unclear or where symptoms were absent from an early age; and to discover novel variants.Sansović et al: Spectrum of genetic variants in 306 patients with non-syndromic hearing loss from Croatia www.cmj.hrGenetic factors cause 60% of congenital hearing impairment, and about 70% of congenital hearing loss (HL) is non-syndromic (1).A major challenge in the study of congenital non-syndromic hearing loss (NSHL) is extreme genetic heterogeneity, whereby the same or very similar clinical phenotype is the result of pathogenic variants in a considerable number of genes.To date, 153 genes have been associated with NSHL, of which 63 are associated with autosomal dominant (AD) loci DFNA (DeaFness, Nonsyndromic, A indicates autosomal dominant inheritance), 86 with autosomal recessive (AR) loci DFNB (DeaFness, Non-syndromic, B indicates autosomal recessive inheritance), seven are sex-linked genes, and nine are mitochondrial associated genes.Nevertheless, different mutations in one gene (for example, GJB2, TECTA, MYO7A) can cause recessive and dominant forms of NSHL, which makes genetic counseling for affected families difficult (2).Pathogenic variants at the locus 13q12, DFNB1 (GJB2 and GJB6 genes), account for about half of all DFNB cases and are the most common cause of severe-to-profound DFNB (3).The gap junction beta-2 (GJB2) gene encodes the protein connexin 26, which is an integral part of gap junctions in the cochlea.Variants in the GJB2 gene show a regional/ ethnic distribution.In European and American white populations, the most common variant responsible for about 70% of DFNB1 is c.35delG.The carrier frequency of this variant is particularly high in the European Mediterranean countries (4,5).
In 30% of hereditary HL cases, HL may be one of the features of about 400 different syndromes.Sometimes the symptoms of these syndromes are very mild, nonspecific, or do not manifest at all at an early age; therefore, it is difficult to distinguish NSHL from the syndromic form of HL (8).
Next-generation sequencing (NGS) strategies, clinical exome sequencing (CES), and whole exome sequencing (WES) have significantly contributed to the diagnosis of genetically and clinically heterogeneous conditions, including hereditary HL.They have increased the diagnostic yield and contributed to the discovery of new genes associated with HL (9).
This study is an update on our previous study on GJB2-related NSHL in Croatia (10).The current study encompassed more than five times the number of participants from the previous study, and was enhanced by the inclusion of CES.The aim was to report the genetic causes of NSHL in a retrospective study of patients referred from several clinical centers and to determine the diagnostic yield using a combination of multiplex ligation-dependent probe amplification method (MLPA) and Sanger sequencing with the more contemporary CES technology.

Patients
In the period between March 2006 and October 2023, 352 unrelated patients with HL from Croatia were referred to our Department of Medical and Laboratory Genetics, Endocrinology and Diabetology, Children's Hospital Zagreb for NSHL genetic testing.Clinical geneticists at University Children's Hospital Zagreb, University Hospital Centre Split, University Hospital Centre Osijek, and University Hospital Centre Zagreb evaluated patients by obtaining family history, and performing physical examination and additional laboratory testing when appropriate.Forty-six unrelated patients with additional features and symptoms suggesting syndromic or acquired HL (such as infection, trauma, noise, ototoxic drugs, and premature birth), those with adult onset HL, unilateral HL, and those with insufficient medical data were excluded from the study.The final sample consisted of 306 patients (169 men [55.2%]) who were diagnosed with childhood familial or sporadic, mild to profound NSHL by a clinical geneticist.Severity of HL was classified as follows: <20 dB, normal hearing; 20-39 dB, mild HL; 40-69 dB, moderate HL; 70-94 dB, severe HL; and >95 dB, profound HL (mean of hearing at 0.5-1-2-4 kHz).The ethnicity of the patients was not recorded because the vast majority were white Eastern Europeans and/or of Slavic origin.The median age of probands at the time of clinical assessment was 3.4 years (41 months, range 1 month-44 years).Informed consent was obtained from all patients or, in the case of minors, from their parents/legal guardians.
MLPA analysis and Sanger sequencing of the GJB2 gene MLPA analysis was performed by using SALSA MLPA kit P163, version B1, C1, and D1 GJB WFS1 (MRC Holland, Amsterdam, The Netherlands), according to the manufacturer's recommendations (11) Holland).A reduced signal of one of the mutation-specific probes can point toward the presence of this mutation or a (partial) deletion of GJB2.If that was the case, Sanger sequencing of the target sequences in exon 2 of GJB2 was performed for confirmation.The exon 1 and flanking donor splice site of the GJB2 gene was amplified with primers described earlier (10).
In all patients in whom MLPA analysis did not show copy number variation, Sanger sequencing of the complete coding region (exon 2) of the GJB2 gene was performed.
The coding region was amplified and sequenced by using primer sequences described elsewhere (12).Sequencing was carried out with a BigDye v1.1 or v3.1 sequencing kit (Applied Biosystems, Thermo Fisher Scientific), followed by capillary electrophoresis on an Applied Biosystems ABI PRISM® 310 or SeqStudio capillary sequencer.

CeS -library preparation, sequencing, and bioinformatics
CES was implemented in our laboratory diagnostic setting in 2018.From April 2019 to October 2023, CES was performed in 21 patients who tested negative for GJB2 biallelic variants.The DNA library for CES analysis was generated with enrichment oligos by using Illumina DNA Prep with Enrichment (Illumina Inc., San Diego, CA, USA) focusing on the exons of 4813 disease-associated genes (TruSight One Panel, Illumina Inc.).Sequencing was performed on Illumina's MiniSeq, MiSeq, and NextSeq platforms in 2 × 150 pair-end reads.Variants were considered for interpretation if a minimum median coverage was 70 × and if they were covered by at least 20 reads.

Variant interpretation
ClinVar, HGMD, LOVD, and Hereditary Hearing Loss Homepage databases were used to search for variants associated with NSHL (13)(14)(15).The clinical significance of the variants was assessed with the use of patho-genicity and conservation scores from VarSome Clinical and Ensembl Variant Effect Predictor.All variants were classified according to the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines (16).Only pathogenic, likely pathogenic variants, and variants of uncertain significance (VUS) were considered as possible causes of NSHL.Common benign variants were not presented in this report because they were not the subject of research.
Clinically relevant variants were filtered according to phenotypes listed in the Human Phenotype Ontology database (HPO): Sensorineural Hearing Impairment (HP:0000407) and Hearing Impairment (HP:0000365).If NSHL was accompanied by some other clinical features, the variants in genes associated with additional HPO terms were also surveyed (17).

Spectrum of variants in the GJB2 gene
Among 306 NSHL patients, 234 disease-associated GJB2 alleles were detected.In total, 19 clinically relevant GJB2 variants were identified, of which 18 were reported as pathogenic or likely pathogenic, and only c.-1G>A variant was categorized as a VUS.All GJB2 variants detected have already been reported in individuals with NSHL (2).The most common variant by far was c.35delG, accounting for 73.5% of mutated alleles.It was followed by alleles c.-23 + 1G>A, c.71G>A, c.109G>A, c.269T>C, c.101T>C, and c.313_326del, whose frequency was 1.3%-5.1%.All other alleles were detected only once or twice.Except c. 551G>A, all alleles were associated with an AR mode of inheritance (Supplement 1, Figure 1).
In six of the 11 probands, a heterozygous variant in the TECTA, NOG, GATA3, SOX10, and COL2A1 genes resulting in AD HL was detected.Compound heterozygous disease-causing variants were identified in five probands in genes associated with AR HL (SLC26A4, PCDH15, TMPRSS3, USH2A, and MYO15A).None of the variants were found in the homozygous state.One female patient had two pathogenic heterozygous variants: one associated with AD syndromic HL and the other associated with the non-HL phenotype.The studied group consisted of five familial (MYO15A, NOG, SLC26A4, and TECTA twice) and six spo-radic cases of HL (TECTA, GATA3, PCDH15, TMPRSS3, USH2A, and SOX10/ COL2A1).
With the exclusion of 13 individuals with monoallelic GJB2 recessive mutations, the diagnostic yield of the molecular testing of the GJB2 variants by MLPA and Sanger sequencing in NSHL patients was 36.3% (111/306).By CES analysis performed on 21 participants, we were able to elucidate the genetic cause of HL in 10 patients (47.6%), without considering one VUS variant in the TECTA gene (Figure 2).(23,24).
In addition to the 35delG, variants c.71G>A, c.109G>A, c.269T>C, c.101T>C were found in a patient with biallelic, as well as in a patient with monoallelic GJB2 variants.These variants were also detected at higher frequencies in other white populations (12,(22)(23)(24)(25).
Using CES, we found the genetic cause for syndromic HL in six probands, and one pathogenic variant causing non-HL phenotype in one patient.In two patients, we found disease-causing AR variants in the USH2A and PCDH15 genes.Similar to other reported cases of Usher syndrome, USH2A and USH1F patients are often initially misdiagnosed with NSHL due to the absence of retinitis pigmentosa in early childhood (8).
In a 9.5-year-old girl with congenital severe bilateral HL, juvenile rheumatoid arthritis, and myopia, a de novo frameshift mutation c.404dup in the GATA3 gene was identified.
Heterozygous mutations in the GATA3 gene are a known cause of Barakat syndrome (MIM#146255).However, the girl, except for HL and asymptomatic hyperthyroidism, did not show any other features of Barakat syndrome (26).Because of hyperthyroidism she was monitored by an endo-  eral HL and mild specific learning and speech disabilities.
The position was less conserved, and the in silico predictions were slightly more in favor of the benign nature of the variant, but this variant is extremely rare in the general population, and the proband`s parents had not yet been tested.Nevertheless, since clinical features of the boy were more suggestive of a non-genetic etiology of HL, we believe that the c.650C>T variant is probably benign.
A likely pathogenic c.6167G>T variant in TECTA was detected in a 9.5-year-old girl with moderate HL.The substitution replaced conserved amino acid cysteine at codon 2056 with phenylalanine in the zona pellucida (ZP) domain of α-tectorin.Missense variants in the ZP domain of α-tectorin predominantly cause mid-frequency HL.This variant was absent from the gnomAD exomes and gno-mAD genomes database, and the multiple in silico predictions were in favor of the pathogenic nature of the variant.The proband`s multiple family members were affected by HL: mother and father, both brothers, three siblings on the mother's side, and the mother's grandmother.Every family member was using hearing aids.Except for the father, all family members had prelingual HL.The audiograms of the proband and other family members with HL were not available.The mode of segregation of the variant in the family is unknown because the family did not undergo genetic testing.
In the NOG gene, the frameshift variant c.291delC was found in a two-year-old girl and her mother.The girl had facial dysmorphism, congenital moderately severe-to-severe bilateral progressive HL, convergent strabismus, amblyopia of the right eye, hypermetropia, short neck, accelerated bone growth, and partial syndactyly of the second and third toe on both feet.Her mother had conductive progressive HL, otosclerosis, hypermetropia, and slightly broader thumbs and big toes.The mother's father also had HL.The phenotype of the proband and her mother corresponds to the features of AD Teunissen-Cremers syndrome (MIM#184460) (28).The variant was categorized as pathogenic since it is a loss-of-function (LOF) variant that does not have a gnomAD exomes and gnomAD genomes entry, the phenotype of the family was highly specific to the above-mentioned syndrome, and there was segregation of the variant in the affected family members.The diagnostic rates of applied genetic methods were comparable with those reported previously (3,33).The reason for the slightly higher detection rate (44%) of pathogenic GJB2 variants in the previous study is probably the sample size bias, that is, the higher representation of patients with congenital, AR severe-to-profound NSHL in a relatively small cohort compared with the current study.We must emphasize that CES analysis covered only 10.8% (21/195) of patients negative for GJB2 biallelic variants.Nevertheless, it allowed us to elucidate the genetic cause of HL in 10 more patients, resulting in a total diagnostic yield of 39.5% in the NSHL cohort (Figure 2.).
This study has some minor limitations.Some participants had insufficient audiological data (eg, audiograms, progression of HL), incomplete family history, and irregular follow-up examinations, due to which we were not able to show the GJB2 genotype-phenotype correlation.It is difficult to recruit some HL-families with multiple members affected (most often both parents) for genetic testing because they do not consider HL a disorder but a special condition that should not be tested or treat-ed as such.Fortunately, with today's cochlear implant options and those offered by gene therapy, more and more people are getting involved in genetic testing for HL.Regarding the methodology, 89.2% (174/195) of GJB2-negative patients were not analyzed by CES.The reasons for this were as follows: 1) the implementation of CES analysis started only in 2018, when some adult patients were no longer followed up in pediatric clinics and 2) the small capacity of MiSeq and MiniSeq genomic analyzers, which were used most of the time in CES.This study did not include copy number variant (CNV) analysis in NSHL genes due to the insufficient number of patients analyzed.Since recent studies have shown that CNVs contributed to the genetic diagnosis of NSHL in 15.2% of patients, CNV analysis, especially of the STRC and OTOA genes, remains to be implemented in further studies (34,35).
In conclusion, all GJB2 variants detected in this study have already been reported in individuals with NSHL, but almost one third of all variants detected by CES were novel.The c.35delG was the most common variant.In the non-GJB2 gene group, the most variants were found in TECTA.With applied methods, we were able to elucidate the genetic cause of HL in 39.5% of patients.CES enabled us to diagnose almost half of the patients with HL (10/21); distinguish NSHL from the syndromic form of HL in cases where the phenotype was indistinctive or symptoms were absent at an early age, such as in retinitis pigmentosa in Usher syndrome; and discover an additional genetic cause of non-HL phenotype in a patient with syndromic HL (WS4C).Nevertheless, considering the high prevalence of GJB2 variants among the tested NSHL Croatian participants, the recommended NSHL testing strategy would still be screening for the GJB2 variants with MLPA and Sanger sequencing, followed by NGS (CES or WES) analysis in patients negative for GJB2-biallelic variants.
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dISCuSSION
FIguRe1. the spectrum and frequency of 234 disease-associated GJB2 alleles detected in 306 unrelated patients with childhoodonset non-syndromic hearing loss.
. The latest version, 124 participants with GJB2 variants, 110 had biallelic GJB2 variants and 14 had only one GJB2 variant.
c.35delG allele, which was present in 90.5% (38/42) of compound heterozygotes.The remaining 14 patients had a single heterozygous mutation.Again, in this group c.35delG was the most frequent allele, followed by c.109G>A and c.71G>A (Table
(18)e this COL2A1 variant is inherited from the patient's father with normal hearing, we assume that this variant is a cause of the ocular phenotype present in both affected family members.The non-syndromic form of Stickler syndrome type I is usually not associated with sensorineural HL.Therefore, a defect in SOX10 alone is very likely responsible for the HL phenotype in the proband.TECTA-related AD NSHL is one of the most frequent subtypes of AD NSHL(6).TECTA is comprised of 23 exons, and more than 80% of missense variants in TECTA submitted in the ClinVar database are of uncertain significance(13).Many of these variants are private, meaning that they occur in a very small number of cases, usually familial.Therefore, it is challenging to decipher missense TECTA variants causing AD NSHL.In this study, we detected two likely pathogenic TECTA variants in families with multiple members affected by moderate-to-severe NSHL.While determining the clinical significance of variants, one should use not only different in silico prediction tools, but also the phenotypes of patients and the study of family segregation(18).